Retinal camera filter for macular pigment measurements

ABSTRACT

Apparatus for use in measuring the density and spatial distribution of macular pigment in an eye comprises a camera ( 4 ) for capturing a colour image of an eye, at least one filter ( 36 ) for filtering light reaching the camera (preferably by filtering the light illuminating the eye). The filter ( 36 ) has a transmission spectrum with one peak in the region of light absorbed by the pigment and another peak in a region where no such absorption occurs. The filter increases the sensitivity of the camera to macular pigment whilst enabling the effect of other pigments to be reduced or eliminated. A method of measuring macular pigment involves obtaining a colour image of an eye, the image having two colour components each having a spectrum having a respective one of said peaks. Corresponding portions of the components are mathematically combined so as to provide a measurement of macular pigment density and the results of the combination are used to provide an output representative of the contribution of macular pigment to the image.

FIELD OF THE INVENTION

This invention relates to apparatus for use in inspecting the densityand spatial distribution of macular pigment in an eye, and to a methodof determining said density and spatial distribution.

BACKGROUND TO THE INVENTION

Macular pigment is a yellow pigment situated in the central portion ofthe human retina. The absorption spectrum for the pigment has a peak forlight of a wavelength of 460 nm and zero for light for a wavelength of540 nm, so that the pigment absorbs significant amounts of the shorterwavelength light, whilst having little or no effect on light of thelonger wavelength.

The highest concentrations of macular pigments are to be found in theregion of the retina which has a very high number density of conereceptors, and is coupled with a disproportionately large area of thevisual cortex, giving that region a high degree of visual acuity.

It has been proposed that the macular pigment protects the retinaagainst harmful effects of short wavelength radiation, and accordinglymuch work has been devoted to measuring the optical density, and spatialdistribution, of macular pigment in various subjects in order todetermine whether there is any correlation between irregularities in theamount of macular pigment present and certain defects.

A flicker photometer is an instrument that enables a subjectivemeasurement of macular pigment density to be made. The flickerphotometer projects green and blue light in an alternating sequence intoa subject's eye, and the subject is able to vary the relative intensityof light of one of those colours until a minimum or no flickering isperceived.

Photographic methods have also been used to obtain an objectiveindication of the macular pigment density/spatial distribution, but inorder to be effective, have involved dilating the subjects pupil,bleaching photo pigments to minimise their contributions and thenphotographing the fundus twice, once in blue light and once in greenlight. Those images are then digitised (if not already captured by a CCDcamera), combined in registration with each other, logarithmicallytransformed and then subtracted.

However, ensuring that the images are precisely registered, is a timeconsuming step which places high demands on image processing softwareand hardware.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is providedapparatus for use in measuring the density and spatial distribution ofmacular pigment in an eye, the apparatus comprising a camera forcapturing a colour image of the retina of an eye under examination,filter means for filtering light reaching camera, the filter meanshaving a transmission spectrum which has a peak in the region of thewavelength of light absorbed by the pigment and another peak in a regionat which no such absorption occurs.

A conventional colour camera can obtain a colour image from a singleexposure, but this image, whilst providing a representation of thecolour of the photographed features, does not have sufficient colourresolution for use in the measurements of macular pigment/spatialdistribution. However, the filter of the present invention increases thesensitivity of the apparatus to said macular pigment since the filterwill pass light having a component at the peak of absorption of themacular pigment and another which will be unaffected by the pigment, sothat the captured image has a component which is greatly affected bymacular pigment density and another, reference component which is not.

Since both components are present in a single image, there is no needfor separate images to be obtained, and the invention therefore alsoavoids the problem of achieving image alignment. In addition, aconventional camera can be used, so that apparatus in accordance withthe invention may be relatively cheap to produce.

In order to provide good resolution, the filter means preferably has atransmission spectrum which is substantially zero between said twopeaks. To that end the transmission spectrum may to advantage not exceed0.001% between said peaks. Preferably each peak is no more than 40 nmwide.

The filter means may be placed anywhere in the path of light whichilluminates the eye and travels to an image capture device, for exampleone or more CCD arrays in the camera. Preferably, however, the filtermeans is situated in between the eye and an illuminating light source,so that the spectrum of light which illuminates the eye has said peaks.Thus, for example, a conventional 3 CCD array retinal camera, whichtypically has a flash lamp and an associated and interchangeable filterfor the flash lamp, can be converted into apparatus according to theinvention, simply by replacing the existing filters with said filtermeans.

Since the advantages of the invention can be achieved by selecting anappropriate spectrum of illuminating light, there is provided, inaccordance with the second aspect of the invention, apparatus for use inthe measuring of the density and spatial distribution of macular pigmentin an eye under examination, the apparatus comprising illumination meansfor illuminating said eye and a camera for capturing a colour image ofthe eye, when so illuminated, wherein the illumination means is operableto illuminate the eye with light the spectrum of which has a first peakat a wavelength of light which is absorbed by the macular pigment and asecond peak at a wavelength at which substantially no such absorptionoccurs.

Preferably, the spectrum of said illuminating light falls tosubstantially zero between these two peaks.

The filter means preferably comprises a single filter having both saidpeaks in its transmission spectrum.

Preferably, one of said peaks is at the wavelength corresponding to bluelight, the other at that corresponding to red light.

Preferably, said first peak is at 460 nm, the second at 600 nm.

The filter may conveniently be a triple bandpass filter, thetransmission spectrum of which has a further peak and a wavelengthcorresponding to green light (e.g. 540 nm).

The filter may be a proprietary item available from, for example, OMEGAOPTICAL.

Preferably, the apparatus includes an image processor for processing theimage captured by the camera, wherein the image processor is programmedto subtract the reference component of the image from the component inthe absorption spectrum of the macular pigment, thereby to remove thecontribution to the image of pigments other than the macular pigment.

Preferably, the processor is operable to display the results of thesubtraction as a macular pigment map.

Preferably, said subtraction is of the logs of the intensities of thetwo components.

Preferably the image processor is operable to take the logs of threeimages, each corresponding to a respective peak of the triple bandpassfilter's transmission spectrum, and to combine these so as to eliminateany contributions from non uniform distributions of both melanin andphotopigments.

If, however, the haemoglobin and melanin are uniformly distributed inthe retina, they will cause a uniform reduction in image intensity,which leaves only three unknown pigment distributions: macular pigment,rod photopigment and cone photopigment.

In this case, the image processor is preferably operable to determine,from the three images, the distributions macular pigment, rodphotopigment and cone photopigment across the retina.

According to a third aspect of the invention, there is provided a methodof measuring macular pigment density and spatial distribution in an eye,the method comprising the steps of,

-   a) capturing a colour image of the retina of the eye, the image    having a first and second colour component, the first colour    component having a spectrum the peak of which is at a wavelength at    which the absorption by macular pigment is at a maximum and a second    peak at which substantially no such absorption occurs;-   b) subtracting one of the image components from the other, at each    region of the image, to remove at least some colour contributions    not arising from the macular pigment and-   c) providing an output representative of the contribution of the    macular pigment to the image.

Preferably the step of capturing the image involves illuminating the eyewith light, the spectrum of which has said first and second peaks.

The image may be captured by means of a camera and a filter which has afirst and second peak its transmission spectrum, corresponding to thetwo peaks of the components, and which filters the light forming theimage captured by the camera. The filter may be in the path of lightfrom the eye under examination to the camera, but is preferably in thepath of light from a source of illumination to the eye.

BRIEF DESCRIPTION OF DRAWINGS

The invention will now be described, by way of example only, withreference to the accompanying drawings in which:

FIG. 1 is an external view of apparatus in accordance with theinvention;

FIG. 2 is a simplified schematic view of optical elements and pathswithin the apparatus;

FIG. 3 is the transmission spectrum of a triple bandpass filter used inthe apparatus;

FIG. 4 is the optical density plot for the triple bandpass filter;

FIG. 5 is a plot of calculated macular pigment density against positionalong a vertical line passing through the fovea in the retina of an eyeunder examination;

FIG. 6 is a surface plot showing calculated macular pigment densityacross a retina;

FIG. 7 is an image of a retina photographed using apparatus according tothe invention; and

FIG. 8 shows a spectral response from 3 CCDs used by the apparatus toprovide an electrical output signal representative of a captured imageof a retina.

DETAILED DESCRIPTION

The camera shown in FIG. 1 is a modified version of a non mydriatricretinal camera, in this case the TOPCON TRC-NW6SF camera. The cameracomprises a housing 1 containing illumination and imaging optics and aflash lamp. At one end of the housing 1 there is an objective lensassembly 2, and at the other end a 3CCD (charge coupled device) camera 4for generating a three component colour output signal representative ofa captured image obtained via the imaging optics in the housing 1. Therear of the housing 1 is also provided with an LCD view finder screen 6,and supports a shutter control 8. Attached to the front of the housing 1is a head support 10 comprising a headband 12 and a chin rest 14. Thehead support 10 locates the head of the subject to facilitate thecorrect positioning of the eye under examination relative to theobjective lens assembly 2.

FIG. 2 shows, in simplified form, the illumination and imaging opticswithin the housing 1, as well as an eye under examination 16, thecamera's flash lamp 18 and a focusing lamp 20. The focusing lamp 20 isused to illuminate the eye 16 while the operator is setting up thecamera to photograph that eye. The illumination provided by the lamp 20enables the image of the retina of eye 16 to be viewed on the viewfinder screen 6 so that the operator can correctly position the eye andfocus the camera. Light from the lamp 20 passes through a focusing lenssystem 22 to a beam splitter 24 in the form of a half silvered mirror,from which it is reflected through a filter assembly 26. The assembly 26comprises a holder 28 which holds four filters, respectively referenced30, 32, 34, and 36, and which is rotatable about an axis parallel to thebeam of light from the focusing lamp 20 to bring any selected one ofthose filters into registry with that beam. It will be appreciated thata holder capable of carrying different numbers (more or fewer) filterscould be used in the camera. In the present case, the filters 30-34 areused for standard retina photography, whilst the filter 36 is a triplebandpass filter, described below, which enables the image captured bythe camera to be used to measure macular pigment density and spatialdistribution on the retina of the eye 16.

Light passing through the filter 36 then passes to an annular mirror 38via a reflecting mirror 40 and focusing lenses 42, 44 and 46. The mirror38 reflects that light via the objective lens assembly 2 into the eye 16to illuminate the retina of that eye. That light is reflected from theretina and some of it passes back through the lens 2 which directs thelight through the aperture (referenced 48) in the mirror 38, through afurther system of lenses 50, 52, 54 and 56 which focus an image of theilluminated retina onto the image plane of the CCD camera 4. The TOPCONTRC-NW6 camera is supplied with a neutral filter for use in normalcolour photography (for example for use in diabetic screening) and anexciter filter for use in fluorescein angiography. These filters may beinterchanged with other filters, and modification to the cameranecessary to convert it into apparatus according to the invention isachieved by replacing one of those filters with the triple bandpassfilter 36. In reality, the camera has a more complex arrangement ofoptical elements than is indicated by FIG. 2, but since these are, savefor the filter 36, identical to those used in the known camera, theyhave not been described in detail.

The output of the camera 4 is connected to a computer 5 which has avideo capture card for enabling the output to be recorded onto thecomputer's hard drive for subsequent processing.

The CCD camera 4 has three CCD arrays and associated red, green and bluecolour filters. Each CCD array is positioned behind a respective one ofthe three filters, and the camera includes a beam splitter forprojecting the image of the retina of the eye 16 onto each of the 3 CCDarrays through its respective filter. The output of each array willtherefore represent an array of grey scale pixel values which itselfconstitutes an intensity map of the filtered light received from theretina. The output of the CCD arrays therefore constitutes red, greenand blue channels.

FIG. 8 illustrates the spectral response of the blue (B) green (G) andred (R) channels in the camera 4. Were white light to be used toilluminate the retina under inspection, the blue green and red channelsof the camera output would not provide sufficient colour resolution toenable macular pigment density to be measured. However, the spectralresponses from the three CCD arrays in the camera 4 will be shaped intonarrower wave bands by the filter 36, since the transmission spectrum ofthis filter has three relatively narrow bands, referenced 50, 52 and 54in FIG. 3, in its transmission spectrum. The width of each of thesebands is considerably narrower than that of the three bands, B, G and R,the transmission spectrum between adjacent bands is substantially zero,as is illustrated in the optical density map of FIG. 4 in which thevertical axis is minus one multiplied by the log (to base ten) of thetransmittance. Thus, the transmittance of the triple bandpass filter 36between the transmission bands does not exceed 0.00001 (i.e an opticaldensity of 5). A filter having these spectral characteristics isavailable from Omega Optical. The interaction between the triplebandpass filter 36 and the filters in the CCD camera 4 is such that, ofthe light transmitted through the filter 36, the light within the band50 will only affect the blue output channel for the camera 4, all lightin the band 52 will affect the green channel whilst light in the band 54only appears in the red channel. Thus, light transmitted in each of thethree bands of the bandpass filter 36 will only affect the output from arespective one of the 3 CCD arrays in the camera 4.

The method of operation of the apparatus, and the analysis of theretinal image captured by the apparatus, will now be described.

Initially, the subject places his or her head against the head support10, and the focusing lamp 20 and camera 4 are activated respectively toilluminate the eye 16 and to capture a video image thereof. That imageis displayed on the display 6 and the operator adjusts the controls ofthe camera to focus and align that image. The manner of this adjustmentis the same as for the known retinal camera on which the presentapparatus is based.

The operator then activates the shutter switch, causing the lamp 18 toflash and a shutter (not shown) in the camera 4 to operate, so that thecamera 4 captures the colour image of the retina of the eye 16 when thelatter is being illuminated by the lamp 18 through the filter 36, i.e.with light having a spectrum corresponding to the transmission spectrumof FIG. 3.

The camera supplies R, G and B signals to the computer 5, said signalsrepresenting an array of grey scale pixel values for each of the 3 CCDarrays.

Image analysis software (for example ImagePro Plus) which has beenpre-installed on the computer 5 is then used to analyse the capturedimage. This is a powerful application capable of performing manyoperations, including those needed to generate an optical density map ofthe macular pigment of the retina. However, it is envisaged that other,simpler software packages could be used to achieve the same end, usingan analysis technique developed from the underlying theory summarisedbelow.

We will assume a general situation of non-uniform illumination of theretina by the camera's flash lamp. Let the incident intensities byI_(F,B), I_(F,R), I_(P,B) and I_(P,R), where the subscripts F and Prefer to a foveal and peripheral retinal location (no macular pigment),and the additional subscripts B and R refer to the blue (460 nm) and redwavelength bands, respectively of the light source (i.e flash lamp 18and filter 36). The analysis would not be affected if the greenwavelength band had been chosen instead of the red. Similarly letR_(F,B), R_(F,R), R_(P,B) and R_(P,R) be the corresponding reflectancesof all retinal layers posterior to the macular pigment. Finally, T isthe 460 nm transmittance of the macular pigment at the foveal location,and the logarithms/log differences in this description are to base ten.

For the blue illumination, the log difference in reflected intensitiesbetween the foveal and peripheral locations will be given by

${{LD}_{B} = {{{\log \; I_{F,B}T^{2}R_{F,B}} - {\log \; I_{P,B}R_{P,B}}} = {\log \frac{I_{F,B}T^{2}R_{F,B}}{I_{P,B}R_{P,B}}}}},$

and for red illumination by

${LD}_{R} = {{{\log \; I_{F,R}R_{F,R}} - {\log \; I_{P,R}R_{P,R}}} = {\log \frac{I_{F,R}R_{F,R}}{I_{P,R}R_{P,R}}}}$

The factor T² in the first equation is due to the double passage of thelight through the macular pigment.

$\begin{matrix}{{Subtracting},{{{LD}_{R} - {LD}_{B}} = {\log \frac{I_{F,R}R_{F,R}I_{P,B}R_{P,B}}{I_{P,R}R_{P,R}I_{F,B}T^{2}R_{F,B}}}}} & (1)\end{matrix}$

The spectral distributions of light on the fovea and periphery will bethe same,

${\therefore\frac{I_{F,R}}{I_{F,B}}} = {\frac{I_{P,R}}{I_{P,B}}.}$

It will also be assumed that the reflectance spectrum is the same ineach location,

${\therefore\frac{R_{F,R}}{R_{F,B}}} = \frac{R_{P,R}}{R_{P,B}}$

Equation (1) then becomes

${{LD}_{R} - {LD}_{B}} = {{\log \frac{1}{T^{2}}} = {2\; D}}$

where D(=−log T) is the optical density of the macular pigment at 460nm. Thus

D=½(LD _(R) −LD _(B))  (2)

Using ImagePro Plus, the spatial distribution of D is obtained from asingle retinal image as follows:

-   1. Individual grayscale images are extracted from the original    image, corresponding to the modified blue and red (and green)    channels of the camera 4.-   2. The greyscale images are transformed to floating point format to    minimise loss of information in the subsequent steps.-   3. The “red” and “blue” images are logarithmically transformed.-   4. The “log blue” image is subtracted from the “log red” image.-   5. The resulting image is halved, in accordance with equation (2).

The result will be a grayscale image, an example of which is shown inFIG. 7, in which the light area 56 is the area of macular pigment. Avariety of options is available for further analysis or presentation.The image may be rendered as a surface plot as in FIG. 6 in which thearea of macular pigment is shown as a “hill” in the centre of the image.A density scan may be made along a line through the fovea, for examplealong horizontal or vertical meridians. An example is shown in FIG. 5.From such a plot, the peak macular pigment optical density will beobtained as the difference between the pixel values at the peak and at aperipheral location, such as 80 above the fovea. Alternatively, acircular “area of interest” corresponding to, say, 1.50 may be defined.The average pixel value along the circular line, or the average pixelvalue within the enclosed area, may be obtained. There is evidence thatflicker photometry determines the macular pigment density at the edge ofthe stimulus rather than the average value over the stimulus area. Thus,if a comparison is to be made between flicker photometry andreflectometry, determining the average pixel value along the circularline may be more appropriate.

The new method offers several advantages over traditional reflectometry,which requires the acquisition of separate blue and green images thatmust be precisely registered with each other. Such alignment is possiblewith ImagePro, but it would be too time-consuming for large-scalescreening. With the proposed procedure, the blue and red images will beextracted from a single image and will be perfectly registered. Also,when separate images are acquired, there is the problem of non-uniformillumination of the retina that may be different in the two images. Ascan be seen in the derivation of equation (2), any non-uniformity is thesame in both images, if these are extracted from a single image, and isself-cancelling.

There remains the question of whether to use a red or green image as thereference image. Either fulfils the requirement of showing zero or nearzero macular pigment optical density. However, the green image shows adarkening in the same region as the macular pigment due to the presenceof long and medium wavelength cone photopigments. To minimise thecontribution of these photopigments, they would normally have to bebleached (approx. 5.6 log Td for approx. 3 minutes) prior to theacquisition of the image. However, with a method in accordance with theinvention a triple bandpass filter 36 with the red transmitting bandcentred at approx. 600 nm is used. At this wavelength, the opticaldensity of the cone photopigments is approximately the same as at 460nm, the centre of the blue transmitting band. This photopigment opticaldensity will contribute equally to the red and blue images and will beeliminated by the subtraction process. At 600 nm, rod photopigmentoptical density is approx. zero, but this is not the case at 460 nm andcould affect the comparison between the foveal and peripheral sites inthe blue image. However, the optical density at 500 nm has beenestimated to be about 0.016 at 7° to 100 from the fovea (Brindley G. S.and Willmer E. N. (1952). The reflexion of light from the macular andperipheral fundus oculi in man. J. Physiol. 116, 350-356). This wouldcorrespond to roughly 0.01 at 460 nm and is comparable with the estimateof “Delori F. C., Goger D. G., Hammond B. R., Snoddlerly D. M., Burns S.A. (2001) Macular pigment density measured by autofluorescencespectrometry: comparison with reflectometry and heterochromatic flickerphotometry. J. Opt. Soc. Am., A, Optics, Image Science, & Vision 18,1212-30. Assuming no rods at the foveal site, macular pigment opticaldensity would be underestimated by only about 2 to 4% in the averagesubject.

Apart from photopigments, melanin and oxyhaemoglobin can potentiallyinfluence macular pigment measurements obtained by reflectometry.Oxyhaemoglobin can probably be ignored because its density is the samein the fovea and periphery (12°). Melanin may pose a problem since ithas been shown to have a non-uniform distribution in the retina, peakingin the macula. Also it has an absorbance spectrum that decreases withincreasing wavelength. Thus the blue image would be the most affected,the green image would be moderately affected, and the red image would beleast affected. This would tend to cause the macular pigment opticaldensity to be overestimated by a factor that would be larger if the redimage is used as the reference rather than the green. In principle, theeffects of melanin can be removed. To achieve this, theory indicatedthat equation (2) would need to be replaced by

D=½(rLD _(R) −LD _(B))  (3)

where r is the ratio of the melanin extinction coefficients at 460 and600 nm (approx. 4). Hence the “log red” image would need to bemultiplied by r prior to subtracting the “log blue” image. However, itshould be noted that equation (3) assumes uniform illumination of theretina and a spectrally flat reflector. In addition, the value D givenby (3) will be affected by any non-uniform distribution of photopigmentacross the retina. By exploiting the green image, as well as the blueand red images, we can eliminate the contributions from non-uniformdistributions of both melanin and photopigments. The appropriateequation for D is then

$\begin{matrix}{D = {\frac{1}{2}\lbrack {{{LD}_{R}\frac{r_{2}{r_{4}( {r_{1} - r_{3}} )}}{ {{r_{1}r_{4}} - {r_{2}r_{3}}} )}} - {LD}_{B} + {{LD}_{G}\frac{r_{1}{r_{3}( {r_{4} - r_{2}} )}}{{r_{1}r_{4}} - {r_{2}r_{3}}}}} \rbrack}} & (4)\end{matrix}$

where the coefficients, r_(n), are the ratios of melanin or photopigmentextinction coefficients at different pairs of wavelengths. Morespecifically the r factors are as follows:r₁=ext. coeff. at the blue wavelength/ext.coeff. at the green wavelengthfor melaninr₂=ext. coeff. at the blue wavelength/ext. coeff. at the red wavelengthfor melaninr₃=ext. coeff. at the blue wavelength/ext. coeff. at the greenwavelength for cone photopigmentr₄=ext.coeff. at the blue wavelength/ext. coeff. at the red wavelengthfor cone photopigment

The ratios are obtainable from the literature. To put equation (4) intopractice, the “log red”, “log green” and “log blue” images will belinearly combined using the appropriate multipliers shown in theequation.

Here, D is the optical density of the macular pigment at the wavelengthof the blue filter band (460 nm) and LD_(R), etc are the logarithmicallytransformed red, green and blue grayscale images. The software (ImageProPlus) is Windows-based and performs each of the following steps.

-   1. Individual grayscale images are extracted from the original    image, corresponding to the filter-modified blue and red and green    channels of the camera.-   2. The “red” “green” and “blue” grayscale images are transformed to    floating point format to minimise loss of information in the    subsequent steps.-   3. The three grayscale images are logarithmically transformed.-   4. The 3 logarithmically transformed images are combined according    to equation (4).

The result is an image of the retina that shows a lighter area (higherintensity/higher pixel value) in the region of the macula. A “value” ofmacular pigment density may be found by taking the average of a set ofpixel values within a circular region (e.g. 1 degree in diameter)centred on the centre of the macula, and subtracting the average of asimilar set centred at a reference location at, say, 8 degrees from thecentre of the macula (where macular pigment density 0). This wouldprovide the average macular pigment density in the central 1 degree.

It will be appreciated that in the maps/plots of FIGS. 5-7, eachindividual pixel represents a mathematical combination of the amounts oflight transmitted through each band of the triple bandpass filter,subsequently reflected from the retina, and modified in the central partof the retina by the transmitting properties of the macular pigment.Thus the macular pigment optical density, D at any point within thiscentral part of the retina is obtained by subtracting from thecorresponding pixel value the pixel value at some non-central retinallocation, such as at an eccentricity of 8°, where macular pigmentdensity is known to be negligible. For example, in FIG. 5, the peakoptical density D is obtained by subtracting from the peak ordinatevalue the ordinate value at pixel number 95, this representing a pointon the retina approximately 8° from the centre of the fovea.

Notwithstanding the above comments on the distribution of rodphotopigments, it is believed that the effect of such pigments on themacular pigment measurement may be eliminated by using an image of theretina illuminated by light at a fourth wavelength. In order to obtainthe second image, the triple bandpass filter 36 is exchanged for afilter with peak transmittance at 680 nm and a bandwidth of 20 nm andthe eye under examination is photographed a second time. The firstphotographs yields the ‘red’ green’ and ‘blue’ images, one from eachrespective CCD array, whilst the second photograph yields a second ‘red’image (at a wavelength longer than that of the first ‘red’ image). Thereare therefore 4 images at difference wavelengths, and these can be usedto obtain the macular pigment optical density in a way which eliminatesthe (small) effect of rod photopigment.

Here, briefly, is how we would obtain the macular pigment opticaldensity distribution, including this new refinement:

-   1. Obtain an image using the triple bandpass filter. Use image    analysis software to extract the grayscale images corresponding to    the red, green and blue channels, as before, and concert these to    logs (LD_(R),LD_(G),LD_(B)).-   2. Obtain a second image using a filter with peak transmittance at    680 nm and a bandwidth of 20 nm, for example. This is a longer    wavelength than the red band of the triple bandpass filter. At 680    nm, the only pigment with a significant absorption is melanin. Again    extract the grayscale image (from the red channel), and convert to    logs, LD_(R).-   3. Use image analysis software to align the LD_(R′) image with the    LD_(R), LD_(G) and LD_(B) images.-   4. Obtain the macular pigment optical density distribution by    combining the 4 images in a linear fashion—

D=−0.525*LD _(B)+0.355*LD _(G)−0.882*LD _(R)+2.60*LD _(R′)

The numerical factors are different combinations of extinctioncoefficients of the 4 pigments at the 4 wavelengths, similar to thoseshown symbolically (4) of the specification.

Since there are four different images and four unknown pigmentdistributions, the cone and rod distributions can also be determinedusing the following equations:

D _(cone)=−0.391*LD _(R)+0.654*LD _(R′)

D _(rod)=0.0254*LD _(B)=−0.355LD _(G)+1.081LD _(R)−0.826*LD _(R′)

1. Apparatus for use in measuring the density and spatial distributionof macular pigment in an eye, the apparatus comprising a camera forcapturing a colour image of the retina of an eye under examination, atleast one filter for filtering light reaching the camera, said at leastone filter having a transmission spectrum which has a peak in the regionof the wavelength of light absorbed by the pigment and another peak in aregion at which no such absorption occurs.
 2. Apparatus according toclaim 1, in which said at least one filter has a transmission spectrumwhich is substantially zero between said two peaks.
 3. Apparatusaccording to claim 2, in which said transmission spectrum does notexceed 0.001% between said peaks.
 4. Apparatus according to claim 1, inwhich each peak is no more than 40 nm wide.
 5. Apparatus according toclaim 1, in which the filter is situated in front of an illuminatinglight source, so that the spectrum of light which illuminates an eyeunder examination has said peaks
 6. Apparatus according to claim 1, inwhich at least one filter is a single filter having both of said peaksin its transmission spectrum.
 7. Apparatus according to claim 1, inwhich one of said peaks is at a wavelength corresponding to blue light,the other at a wavelength corresponding to red light.
 8. Apparatusaccording to claim 7, in which said first peak is at 460 nm, and saidsecond peak is at 600 nm.
 9. Apparatus according to claim 7, in whichthe filter is a triple bandpass filter, the transmission spectrum ofwhich has a further peak at a wavelength corresponding to green light.10. Apparatus for use in the measuring of the density and spatialdistribution of macular pigment in an eye under examination, theapparatus comprising an illumination device for illuminating said eyeand a camera for capturing a colour image of the eye, when soilluminated, wherein the illumination device is operable to illuminatethe eye with light the spectrum of which has a first peak at awavelength of light which is absorbed by the macular pigment and asecond peak at a wavelength at which substantially no such absorptionoccurs.
 11. Apparatus according to claim 10, in which the spectrum ofsaid illuminating light falls to substantially zero between these twopeaks.
 12. Apparatus according to claim 10, in which the illuminatingdevice comprises a light source and a filter having both said peaks inits transmission spectrum.
 13. Apparatus according to claim 10, in whichone of said peaks is at the wavelength corresponding to blue light, theother at a wavelength corresponding to red light.
 14. Apparatusaccording to claim 13, in which said first peak is at 460 nm, the secondat 600 nm.
 15. Apparatus according to claim 12, in which the filter is atriple bandpass filter, the transmission spectrum of which has a furtherpeak at a wavelength corresponding to green light.
 16. Apparatusaccording to claim 10, in which the apparatus includes an imageprocessor for processing the image captured by the camera, wherein theimage processor is programmed to subtract the reference component of theimage from the component in the absorption spectrum of the macularpigment, thereby to remove the contribution to the image of pigmentsother than the macular pigment.
 17. Apparatus according to claim 16, inwhich the processor is operable to generate an output signal in whichthe results of the subtraction are represented as a macular pigment map.18. Apparatus according to claim 16, in which said subtraction is of thelogs of the intensities of the two components.
 19. Apparatus accordingto claim 15, in which the image processor is operable to take the logsof three images, each corresponding to a respective peak of the triplebandpass filter's transmission spectrum, and to combine these so as toeliminate any contributions from non uniform distributions of bothmelanin and photopigments.
 20. A method of measuring macular pigmentdensity and spatial distribution in an eye, the method comprising thesteps of, a) capturing a colour image of the retina of the eye, theimage having a first and second colour component, the first colourcomponent having a spectrum the peak of which is at a wavelength atwhich the absorption by macular pigment is at a maximum and a secondpeak at which substantially no such absorption occurs; b) mathematicallycombining the image components at each region of the image, to remove atleast some colour contributions not arising from the macular pigment andc) providing an output representative of the contribution of the macularpigment to the image.
 21. A method according to claim 20, in which thestep of capturing the image involves illuminating the eye with light,the spectrum of which has said first and second peaks.
 22. A methodaccording to claim 20, in which the image is captured by means of acamera and a filter which has a first and second peak its transmissionspectrum, corresponding to the two peaks of the components, and whichfilters the light forming the image captured by the camera.
 23. A methodaccording to claim 22, in which the filter is situated in the path oflight from a source of illumination to the eye.
 24. A method accordingto claim 20, in which said step of mathematically combining thecomponents comprises taking the logarithms of the intensities of thecomponent at each said regions, and subtracting one logarithm from theother.
 25. A method according to claim 20, in which said image has threecolour components, and said mathematical combination comprisesmathematically combining logarithms for three components in a linearfashion with weighting factors applied to at least one logarithm, so asto eliminate contributions from non uniform distributions of melanin andcone photopigments over the retina.
 26. A method according to claim 25,in which a further image component of the retina is obtained at a fourthwavelength of light, and data on the further image is used in saidmathematical combination to eliminate the effect of non uniformdistribution of rod photopigment across the retina.
 27. A methodaccording to claim 26, in which the further image component is obtainedby capturing a further image of the retina.
 28. A method according toclaim 27, in which the peaks of the spectra of the components aresubstantially at 460 nm (blue), 530 nm (green) 600 nm (red) and 680 nm(far red) respectively.
 29. A method according to claim 28 in which thelogarithms are combined to obtain the macular pigment density, D, at agiven location by the formula:D=−0.525*LD _(B)+0.355*LD _(G)−0.882*LD _(R)+2.60*LD _(R′) where LD_(B),LD_(G), LD_(R) and LD_(R′) are the logarithms of the differences betweenthe intensities at the given location and a peripheral location in theblue, green, red and far red component respectively.